Mechanical rigidity of the liposome membrane is often defined by the membrane bending modulus and is one of the determinants of liposome stability, but the quantitative experimental data are still limited to a few kinds of liposomes. Here, we used atomic force microscopy to investigate the membrane bending moduli of liposomes by immobilizing them on bovine serum albumin-coated glass in aqueous medium. The following lipids were used for liposome preparation: egg yolk phosphatidylcholine, dioleoylphosphatidylcholine, hydrogenated soybean phosphatidylcholine, dipalmitoylphosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane, cholesterol, and N-(carbonylmethoxypoly(ethylene glycol) 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine. By using liposomes of various compositions, we showed that the thermodynamic phase state of the membrane rather than the electric potential or liposome surface modification with poly(ethylene glycol) is the predominant determinant of the bending modulus, which decreased in the following order: solid ordered > liquid ordered > liquid disordered. By using the generalized polarization value of the Laurdan fluorescent probe, we investigated membrane rigidity in terms of membrane fluidity. Atomic force microscopic analysis was superior to the Laurdan method, especially in evaluating the membrane rigidity of liposomes containing hydrogenated soybean phosphatidylcholine and cholesterol. Positively charged liposomes with a large bending modulus were taken up by cells more efficiently than those with a small bending modulus. These findings offer a quantitative method of analyzing the membrane rigidity of nanosized liposomes with different lipid compositions and will contribute to the control of liposome stability and cellular uptake efficiency of liposomal formulations intended for clinical use.

Exosomes mediate communication between cells in the body by the incorporation and transfer of biological materials. To design an artificial liposome, which would mimic the lipid composition and physicochemical characteristics of naturally occurring exosomes, we first studied the physicochemical properties of exosomes secreted from HepG2 cells. The exosome stiffness obtained by atomic force microscopy was moderate. Some liposomes were then fabricated to mimic the representative reported lipid composition of exosomes. Their physicochemical properties and cellular internalization efficiencies were investigated to optimize the cellular internalization efficiency of the liposomes. A favorable internalization efficiency was obtained by incubating HeLa cells with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol (Chol)/1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) (40/40/20 mol %) liposomes, which have a similar stiffness and zeta potential to exosomes. A dramatic increase in internalization efficiency was demonstrated by adding DOPS to simple DSPC/Chol liposomes. We found that DOPS had a more desirable effect on cellular internalization than its saturated lipid counterpart, 1,2-distearoyl-sn-glycero-3-phospho-l-serine. Furthermore, it was shown that the phosphatidylserine-binding protein, T-cell immunoglobulin mucin protein 4, was largely involved in the intracellular transfer of DSPC/Chol/DOPS liposomes. Thus, DOPS was a key lipid to provide the appropriate stiffness, zeta potential, and membrane surface affinity of the resulting liposome. Our results may help develop efficient drug carriers aiming to internalize active substances into cells.

Effective penetration of drug-carrying nanoparticles into solid tumors is a major challenge in cancer therapy. Exploration of the physicochemical properties of nanoparticles that affect penetration efficiency is required to achieve maximum therapeutic effects. Here, we used confocal laser scanning microscopy to evaluate the efficiencies of penetration of fluorescently labeled liposomes into three-dimensional spheroids composed of HeLa cells. The prepared liposomes were composed of phosphatidylcholines and varying contents of cholesterol and/or a polyethylene glycol-modified phospholipid. We demonstrated that the efficiency of penetration into spheroids increased with the bending modulus (i.e., membrane rigidity) of the liposome, as determined by atomic force microscopy (correlation coefficient, 0.84). To clarify the mechanism by which membrane rigidity contributes to the penetration behavior of liposomes, we also analyzed the cellular uptake using monolayer cells. We showed that penetration efficiency was explained partially by cellular uptake efficiency, but that other factors such as liposome diffusion efficiency in the intercellular space of tumor spheroids contributed. Our results quantitatively demonstrate that the bending modulus of the liposomal membrane is a major determinant of liposomal penetration into three-dimensional spheroids. The present study will contribute to the understanding and control of tumor penetration of liposomal formulations.

Nanodiscs are composed of scaffold protein or peptide such as apolipoprotein A-I (apoA-I) and phospholipids. Although peptide-based nanodiscs have an advantage to modulate the size of nanodiscs by changing phospholipid/peptide ratios, they are usually less stable than apoA-I-based nanodiscs. In this study, we designed a novel nanodisc scaffold peptide (NSP) that has proline-punctuated bihelical amphipathic structure based on apoA-I mimetic peptides. NSP formed α-helical structure on 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) nanodiscs prepared by cholate dialysis method. Dynamic light scattering measurements demonstrated that diameters of NSP nanodiscs vary depending upon POPC/NSP ratios. Comparison of thermal unfolding of nanodiscs monitored by circular dichroism measurements demonstrated that NSP forms much more stable nanodiscs with POPC than monohelical peptide, 4F, exhibiting comparable stability to apoA-I-POPC nanodiscs. Intrinsic Trp fluorescence measurements showed that Trp residues of NSP exhibit more hydrophobic environment than that of 4 F on nanodiscs, suggesting the stronger interaction of NSP with phospholipids. Thus, the bihelical structure of NSP appears to increase the stability of nanodiscs because of the enhanced interaction of peptides with phospholipids. In addition, NSP as well as 4F spontaneously solubilized POPC vesicles into nanodiscs without using detergent. These results indicate that bihelical NSP forms nanodiscs with comparable stability to apoA-I and has an ability to control the size of nanodiscs simply by changing phospholipid/peptide ratios.